A cyclodextrin glucosyltransferase with improved product specificity and preparation method thereof

A technology of glucosyl and transferase, applied in the fields of genetic engineering and enzyme engineering, can solve the problems of low efficiency of long-chain glycosyl genistein, and achieve the effect of being beneficial to industrial production

Active Publication Date: 2022-02-15
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, due to the low efficiency of cyclodextrin glucosyltransferase (CGTase) in synthesizing long-chain glycosyl genistein, it is urgent to find a method to improve the synthesis efficiency of cyclodextrin glucosyltransferase for long-chain glycosylated genistein To promote the rapid development of industries related to genistein glycosyl derivatives

Method used

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  • A cyclodextrin glucosyltransferase with improved product specificity and preparation method thereof
  • A cyclodextrin glucosyltransferase with improved product specificity and preparation method thereof
  • A cyclodextrin glucosyltransferase with improved product specificity and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1: Cyclodextrin glucosyltransferase mutants L174P, L174Y or L174D with high genistein glycosylation efficiency

[0027] Cyclodextrin glucosyltransferase mutants L174P, L174Y or L174D are compared with cyclodextrin glucosyltransferase (GenBank JX412224) whose amino acid sequence is shown in SEQ ID NO.1, the 174th leucine is mutated into pro amino acid, tyrosine or aspartic acid. Mutations can be accomplished by chemical total synthesis or PCR.

Embodiment 2

[0028] Example 2: Preparation method of cyclodextrin glucosyltransferase mutants L174P, L174Y or L174D with high genistein glycosylation efficiency

[0029] Chemical synthesis coding amino acid sequence is as shown in the gene of cyclodextrin glucosyltransferase shown in SEQ ID NO.1 (the nucleotide sequence of gene is as shown in SEQ ID NO.2); The gene obtained and pET-20b (+ ) plasmids were ligated after double enzyme digestion (NcoI and XhoI), transformed into Escherichia coli JM109, the transformed product was spread on LB solid medium, cultivated at 37°C for 8h, picked transformants on LB solid medium, and inserted into Cultured in LB liquid medium, cultured at 37°C for 10 hours, extracted the plasmid, sequenced the plasmid, and obtained the recombinant plasmid pET20b-CGT with correct sequencing; transformed the recombinant plasmid pET20b-CG with correct sequencing into Escherichia coli E.coli BL21(DE3 ) to obtain recombinant Escherichia coli pET20b-CGT / E.coli BL21.

[00...

Embodiment 3

[0043] Example 3: Enzyme activity analysis and genistein glycosylation efficiency analysis of cyclodextrin glucosyltransferase mutants L174P, L174Y or L174D

[0044] 1. Enzyme activity analysis

[0045] 1.1. α-cyclization activity

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Abstract

The invention relates to a cyclodextrin glucosyltransferase with improved product specificity and a preparation method, belonging to the fields of genetic engineering and enzyme engineering. The present invention provides three cyclodextrin glucosyltransferase mutants L174P, L174Y and L174D with high genistein glycosylation efficiency. Compared with wild type cyclodextrin glucosyltransferase, they use maltodextrin as sugar When using the base donor to produce glycosylated genistein, the synthesis yield of long-chain glycosylated genistein increased by 0.7, 1.2 and 1.8 times, respectively, which is more conducive to the industrial production of glycosylated genistein.

Description

technical field [0001] The invention relates to a cyclodextrin glucosyltransferase with improved product specificity and a preparation method, belonging to the fields of genetic engineering and enzyme engineering. Background technique [0002] Genistein (also known as genistein, genistein, etc.) is considered to be a soybean isoflavone with the highest activity and function. In legumes, genistein often exists in the form of its glucoside derivative, genistin (also known as 4',5,7-trihydroxyisoflavone-7-glycoside). Genistein has a wide range of pharmacological effects in human and animal cells. The main performances are as follows: 1) It has the effect of chemical anti-cancer (breast cancer and prostate cancer, etc.). Genistein has estrogen-like and anti-hormone effects, can inhibit the activity of related enzymes in the synthesis process of tumor cells, inhibit tumor angiogenesis during the formation of tumor cells, and delay or prevent tumors from becoming cancer cells. ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/10C12N15/54C12N15/70C12N1/21C12P19/60C12R1/19
CPCC12N9/1074C12N15/70C12P19/60C12Y204/01019
Inventor 韩瑞枝倪晔柴宝成姚栋董晋军许国超
Owner JIANGNAN UNIV
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